PROJECT SUMMARY/ABSTRACT In the developing human brain, microglia are a molecularly and morphologically heterogeneous population, interacting with different cell types and undergoing complex maturation programs in a region-specific manner. However, little is known about microglial maturation trajectories and how they are shaped by niche-specific intercellular interactions, hindering our understanding of their role in homeostasis and disease. Based on our preliminary finding that microglia in the developing brain exhibit transcriptional heterogeneity that defines microglia subpopulations, we propose that individual microglia subtypes elicit distinct functional and molecular responses on other cell types in the brain. Using single-cell RNA sequencing (scRNA-seq) in combination with live cell imaging and light-sheet microscopy, we will determine region-specific and age-dependent transcriptional signatures of developing microglia and address the long- standing question of whether these differences are determined by cell-intrinsic programs, or dictated by age- and microenvironment. We will address these questions in the following aims: Aim 1: Identify and validate region-specific molecular trajectories of microglia maturation in the developing human brain. We will identify regionally distinct maturation markers by examining gene expression differences in microglia from major anatomical regions, and then validate these markers using immunohistochemical and morphological analysis in primary tissue samples with light-sheet microscopy. Aim 2: Determine whether microglial regional identity affects brain development in a niche-dependent manner. We will perform heterotopic transplantations followed by unbiased scRNA-seq analysis to study the molecular consequences of microglia interactions with other cell types in a niche-specific manner. This work will achieve the following: (1) provide comprehensive transcriptional profiles of microglia across developmental stages and major anatomical regions in the developing human brain; (2) identify novel markers of microglial maturation and regionalization; (3) determine whether microglia are affected by the cell type composition of their microenvironment in a region-specific manner. Together, these findings will lead to a better understanding of microglial heterogeneity, help to refine existing iPSC-derived microglia models, and provide insight into how perturbations of microglia during development might contribute to neurological disorders such as autism and schizophrenia years later.